Curr. Issues Mol. Biol., Volume 12, Issue 1 (January 2010) – 4 articles

Azo dyes are commonly used in many commercial industries. Some of the azo dyes can produce carcinogenic compounds after being metabolized by azoreductase. Several human intestinal microbiota possess azoreductase activity which plays an important role in the toxicity and mutagenicity of these azo dye compounds. The acpD gene product (AzoEf1) responsible for the azoreductase activity of Enterococcus faecium, an intestinal bacterium, was heterologously expressed, purified and characterized. The protein sequence shares 67% identity with the azoreductase from Enterococcus faecalis, AzoA. Although AzoEf1 possesses many commonalities with AzoA, there are differences in coenzyme preference, residues associated with FMN binding, substrate specificity, and specific activity. AzoEf1 utilized both NADH and NADPH for the reduction of azo dyes, and it contains a leucyl residue at position 104 and threonyl residue at position 19 which differ from AzoA at the active site. Its specific activity was 5095 μM/min/mg and its catalytic efficiency for Methyl red reduction was lower than AzoA. Full article

Numerous bacterial proteins exert their function outside the prokaryotic cell. To this end, both Gram-negative and Gram-positive bacteria have evolved specialized mechanisms to transport their proteins to the bacterial supernatant or host cell cytoplasm, so called secretion systems. These different strategies will be briefly discussed, followed by an in depth description of the Type III secretion system, an efficient molecular syringe assisting Gram-negative bacteria in entrance, growth and survival in eukaryotic host cells. Topics addressed include classification and role of multiple Type III secretion systems, the mechanism of protein translocation into the host cell as well as substrate recognition and chaperoning. Chlamydiales have also been found to encode a Type III secretion system and associated effector proteins. In contrast to the genetic organization in other bacteria, the encoding genes are scattered throughout the genome. To date, no structural information is available on the chlamydial Type III secretion system. We therefore propose a model of the chlamydial Type III secretion system and summarize current knowledge on the role of Type III secretion in the different stages of the chlamydial developmental cycle. Full article

This article describes a one-step procedure based on Taq polymerase for the precise assembly of DNA fragments into circular constructs as long as 6 kb. The only prior step needed was the amplification of the gene to be cloned and the linear vector backbone, and the whole process up to assembly and circularization lasted only 2 days, compared with the conventional method's 2 weeks. Furthermore, the final DNA construct was used to transform Escherichia coli directly without any further treatment. By circumventing the need for DNA ligase, our "Quick Assemble" method offers an improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-directed mutagenesis and whole-DNA library gene shuffling, as well as the construction of new plasmids with any promoter, resistance gene marker, restriction site, or any DNA tag. Full article

Coeliac disease (CD) development involves genetic (HLA-DQ2/DQ8) and environmental factors. Herein, the influence of the HLA-DQ genotype on the gut colonization process of breast-fed children was determined. A cohort of 20 newborns, with at least one first-degree relative with CD, were classified according to their HLA-DQ genotype into high, intermediate and low genetic risk groups, showing 24-28%, 7-8% and less than 1% probability to develop CD, respectively. Faecal microbiota was analysed at 7 days, 1 and 4 months of children's age by fluorescence in situ hybridization. When considering all data, Gram-negative bacteria and Bacteroides-Prevotella group proportions were higher (P<0.05) in the high than in the intermediate and low genetic risk groups. E. coli, Streptococcus-Lactococcus, E. rectale-C. coccoides, sulphate-reducing bacteria, C. lituseburense and C. histolyticum group proportions were also significantly higher (P<0.05) in the high than in the low genetic risk group. Correlations between these bacterial groups and the genetic risk were also detected (P<0.05). In addition, the number and type of CD relative seemed to influence (P<0.050) these bacterial proportions in children at CD risk. At 4 months of age, similar relationships were established between the high genetic risk to develop CD and the proportions of Streptococcus-Lactococcus (P<0.05), E. rectale-C. coccoides (P<0.05), C. lituseburense (P<0.05), C. histolyticum (P<0.05), Bacteroides-Prevotella (P<0.10) groups and total Gram-negative bacteria (P<0.05). The results suggest a relationship between HLA-DQ genes and the gut microbial colonization process that could lead to a change in the way this disorder is investigated. Full article